11 research outputs found

    RML prion infectivity following digestion with 1 mg/ml pronase E.

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    <p>10% (w/v) RML brain homogenate was incubated with pronase E (1 mg/ml at 37°C) for varying incubation times. For each time point RML prion infectivity was measured by the Scrapie Cell Assay and expressed as a percentage of total infectivity present in the untreated sample; mean ± S.E.M. (<i>n</i> = 5).</p

    Pronase E digested and NaPTA precipitated mouse brain homogenate evaluated by silver stain SDS-PAGE.

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    <p>10% (w/v) homogenates from normal CD-1 brain (CD-1) or RML prion-infected brain (RML) were either untreated (Pro −, NaPTA −), digested by pronase E at 100 µg/ml, 37°C for 30 min (Pro +, NaPTA −), precipitated by NaPTA (Pro −, NaPTA +) or sequentially digested by pronase E at 100 µg/ml, 37°C for 30 min and precipitated by NaPTA (Pro +, NaPTA +). The equivalent of 2 µl 10% (w/v) brain homogenate was loaded in each lane and the gel was stained with silver nitrate.</p

    RML prion infectivity and PrP content following digestion with PK, thermolysin or pronase E.

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    <p>10% (w/v) RML prion-infected brain homogenate was incubated at 37°C with (A) PK (50 µg/ml); (B) thermolysin (100 µg/ml) or (C) pronase E (100 µg/ml) for varying incubation times. For each time point, PrP (filled circles) was measured by ELISA and expressed as a percentage of total PrP present in the untreated sample; mean ± S.E.M. (<i>n</i> = 3, PK and thermolysin; <i>n</i> = 5, pronase E). RML prion infectivity (open circles) was measured by the Scrapie Cell Assay and expressed as a percentage of total infectivity present in the untreated sample; mean ± S.E.M. (<i>n</i> = 3, PK and thermolysin; <i>n</i> = 5, pronase E). Immunoblots of the corresponding protease and incubation time point are shown in panels to the right. Blots were probed with anti-PrP monoclonal antibody ICSM35. Apparent molecular masses are shown in kDa. Data in panels A and B have been published previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015679#pone.0015679-Cronier1" target="_blank">[25]</a>.</p

    Pronase E-resistant RML prion infectivity is precipitated by NaPTA.

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    <p>10% (w/v) homogenates from normal CD-1 brain (CD-1, white bars) or RML prion-infected brain (RML, black bars) were either untreated (whole homogenate), incubated at 37°C for 90 min and 20°C for 30 min (temperature control), digested with pronase E at 100 µg/ml, 37°C for 30 min (pronase), precipitated by NaPTA (NaPTA) or sequentially digested by pronase E at 100 µg/ml, 37°C for 30 min and precipitated by NaPTA (pronase + NaPTA). The concentration of PrP in all samples was measured by ELISA (A) and is expressed as a percentage of the total PrP present in the untreated RML samples; mean ± S.E.M. (<i>n</i> = 5). RML prion infectivity was measured by the Scrapie Cell Assay (B) and expressed as total infectivity present in the samples; mean ± S.E.M. (<i>n</i> = 5).</p

    Western blots of PrP<sup>Sc</sup> from infected mouse brains.

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    <p>10% w/v brain homogenates (n = 3 per group) were digested with proteinase K and immunoblotted with anti-PrP monoclonal antibody ICSM35 (D-Gen Ltd, UK). (A) Transmission of Chandler/RML prions to <i>Sod1<sup>−/−</sup></i> and <i>Sod1<sup>+/+</sup></i> (WT) litter mate controls. (B) Transmission of ME7 prion strain to <i>Sod1<sup>−/−</sup></i> and <i>Sod1<sup>+/+</sup></i> (WT) litter mate controls. (C) Transmission of MRC2 mouse adapted BSE prion strain to <i>Sod1<sup>−/−</sup></i> and <i>Sod1<sup>+/+</sup></i> (WT) litter mate controls. No differences were seen between the two groups regardless of prion strain.</p

    Major strain distribution patterns genotyped for HS mice.

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    <p>The number of SNPs is taken from genomic sequence generated by the Sanger Institute (<a href="http://www.sanger.ac.uk/cgi-bin/modelorgs/mousegenomes/snps.pl" target="_blank">www.sanger.ac.uk/cgi-bin/modelorgs/mousegenomes/snps.pl</a>) and spans 5 kb upstream of the 5′UTR start site to the end of the 3′UTR (NCBI Build 37). Ambiguous SNPs have been excluded. The strain distribution pattern (BALB, CBA); (A, AKR, C3H, C57, DBA, LP) was also seen for <i>App</i> (239/978) but this was not genotyped in the HS mice. Other individual strain distribution patterns were seen ≤5 times.</p

    Kaplan-Meier survival curves.

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    <p>Data are shown as % of animals (female) surviving (y-axis) plotted against the number of days post inoculation (x-axis). (A) Transmission of Chandler/RML prion strain to <i>Sod1<sup>−/−</sup></i> and <i>Sod1<sup>+/+</sup></i> (WT) litter mate controls (B) Transmission of ME7 prion strain to <i>Sod1<sup>−/−</sup></i> and <i>Sod1<sup>+/+</sup></i> (WT). (C) Transmission of MRC2 mouse adapted BSE prion strain to <i>Sod1<sup>−/−</sup></i> and <i>Sod1<sup>+/+</sup></i> (WT). A reduction in mean incubation time of 20%, 13%, and 24% was seen in A-C respectively. This reduction in survival was statistically significant for each transmission (P<0.001, Kaplan-Meier log-rank survival test).</p

    Quantification of Sod and PrP<sup>C</sup>

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    <p>. 10% weight/volume brain homogenates immunoblotted with rabbit polyclonal anti-human SOD1 (Abcam) and detected with IRDye800CW (green) conjugated goat anti-rabbit IgG (Li-Cor). Anti-β-actin mouse monoclonal antibody (Sigma) was also included as a loading control and was detected using IRDye680 (red) conjugated goat anti-mouse IgG (Li-Cor). Fluorescence was visualised using an Odyssey infra-red imager (Li-Cor). (A) Brains from <i>Sod1<sup>+/+</sup></i> wild type mice inoculated with PBS compared with end stage Sod1<sup>+/+</sup> wild type mice inoculated with RML, ME7 and MRC2 prion strains. No differences were seen between the groups. (B) Uninfected mice. (C) Total SOD enzymatic activity in 10% (w/v) <i>Sod1<sup>+/+</sup></i> (WT) brains. Brains from terminally sick mice infected with RML, ME7 and MRC2 were compared with uninfected mice. Samples were run in triplicate with n = 6 for each group. Data are shown normalised by total protein content (µg/ml) as determined by a Bradford protein assay (mean ± standard deviation). No significant difference was seen between the groups. (D) PrP<sup>c</sup> levels in <i>Sod1<sup>−/−</sup></i> and <i>Sod1<sup>+/+</sup></i> (WT) litter mate control mice by ELISA. PrP<sup>c</sup> levels (µg/ml) were determined in triplicate using 10% (weight/volume) brain homogenate for <i>Sod1<sup>−/−</sup></i> (n = 3) and <i>Sod1<sup>+/+</sup></i> (n = 3) in a PrP specific ELISA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054454#pone.0054454-Wadsworth2" target="_blank">[32]</a>. Data are shown normalised by total protein content (µg/ml and x 1000) as determined by a BCA assay (mean ± standard deviation). No significant difference was seen between the two groups.</p

    <i>Sod1</i> expression in mouse brain.

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    <p>Panel A and B. Quantification of <i>Sod1</i> mRNA expression in whole brain by real-time RT-PCR. N = 6 for all groups and samples were run in triplicate. All samples were duplexed for <i>Sod1</i> (Fam-label) and an endogenous control <i>GAPDH, β-actin</i> or <i>Thy-1</i> (Vic-label). Expression level is expressed in arbitrary units as normalised by the geometric mean of the quantity of the endogenous controls (<i>y</i>-axis). Error bars represent the standard deviation. (A) <i>Sod1</i> mRNA expression level for parental strain of the HS mice (except LP). (B) <i>Sod1</i> mRNA expression level grouped by allele (A/G) (A = A, BALB, C3H, C57; G = AKR, CBA, DBA). No significant difference was observed between the groups. Panels (C) and (D). Quantification of Sod1 protein in whole brain (10% homogenate, weight/volume) from n = 3 A allele mice (C57Bl/6) and G allele mice (FVB/N). FVB/N mice were used to represent a G allele strain rather than an HS parental strain due to availability of tissue. Samples were immunoblotted with rabbit polyclonal anti-human SOD1(Abcam) and detected with IRDye800CW (green) conjugated goat anti-rabbit IgG (Li-Cor). Anti-β-actin mouse monoclonal antibody (Sigma) was also included as a loading control and was detected using IRDye680 (red) conjugated goat anti-mouse IgG (Li-Cor). Fluorescence was visualised using an Odyssey infra-red imager (Li-Cor). (C) Uninoculated mice, (D) Terminally sick Chandler/RML prion inoculated mice.</p

    SNP genotyping in HS mice.

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    <p>Genomic location is given based on mouse genome assembly NCBI build 37. Details of the <i>Sod1</i> and <i>Il1-r1</i> SNPs are available from the Sanger Centre (<a href="http://www.sanger.ac.uk/cgi-bin/modelorgs/mousegenomes/snps.pl" target="_blank">www.sanger.ac.uk/cgi-bin/modelorgs/mousegenomes/snps.pl</a>). All polymorphisms were analysed by allele discrimination using a 7500 Fast real time PCR system (Applied Biosystems). For probe details see Table S1. For all genotypes, the statistical test used was the Kruskal-Wallis non-parametric ANOVA. The allelic test used was the Mann-Whitney test.</p
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